Direct but flimsy evidence for oomycete effector translocation
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Direct but flimsy evidence for oomycete effector translocation
This page will be used to post published data showing translocation of external oomycete effector into the host cytoplasm
Curated by Mark Farman
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Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible | Direct but flimsy evidence for oomycete effector translocation | Scoop.it

Image from Tyler et al. 2012, MPMI. http://dx.doi.org/10.1094/MPMI-02-13-0051-IA.   Fig. 2. Specific microbe-independent entry of Avr1bNt proteins into soybean root cells and wheat leaf cells. A-E, Soybean root entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488, and counter stained with propidium iodide and 4',6-diamidino-2-phenylindole (DAPI). F-J, Soybean root entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488, mixed with Avr1b N-terminus with RFLR->qFLR mutation labeled with DyLight550, and counter stained with DAPI. A,F, light image; B,G, Dylight488 image; C, propidium image; H, Dylight550 image; D,I,DAPI image; E,J, overlay of the three fluorescent images. In A-J, purified fusion proteins (0.4 mg/ml each) were incubated with soybean (Williams) root tips, for 2 hours at 25oC in PBS buffer adjusted to pH 7.2, then washed for 15 min in formalin (PBS + 10% formaldehyde) containing 0.2 g/ml propidium iodide (A-E only) and 0.4 g/ml DAPI. K-P, Wheat leaf cell entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488 mixed with Avr1b N-terminus (residues 1-50) with RFLR->qFLR mutation labeled by Dylight550. Purified fusion proteins (0.4 mg/ml each) in PBS buffer adjusted to pH 7.2 were infiltrated with a blunt syringe into ~9 day old wheat seedling leaves. Leaves were imaged after 6 h without washing. K, chloroplast fluorescence (excitation 488 nm; emission meta filter 675-715 nm); L, Dylight550 image (excitation 543 nm; emission window 585-615 nm; gain 600 to 654; digital offset -0.1 to 0); M, Dylight488 image (excitation 488 nm; emission window 505-530 nm; gain 580 to 610; digital offset -0.1 to 0); N, overlay of K-M; O, light image; overlay of N and O. Labeling of proteins with Dylight dyes was as described (Sun et al., 2013). The experiments shown in this figure were conducted at Virginia Tech using a Zeiss LSM 510 Meta confocal microscope.

 

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Brett Tyler's comment, April 26, 2013 6:45 PM
Mark, I'm surprised you haven't retracted these comments following our email exchanges. So I'm posting my rebuttal here. First, while leaf epidermal cells do have large vacuoles, root meristematic zone cells, and leaf mesophyll cells do NOT. This information is available in any plant cell biology text book, e.g. Biology 4th edition Ladiges et al. 2010. With regard to the vacuole, there is no evidence whatsoever for a continuous soluble phase connection between the apoplast and vacuole. This fact can also be found in the same text books. With regard to plasmolysis, Avr1bNt-GFP plasmolysis data were shown in Figure S1 panel O of Kale et al (Cell, 142(2), 284–295 2010). Similar plasmolysis data were shown for the fungal RxLR-like effector Ps87 in Figure 2 panel I of Gu et al (PLoS ONE 6(11), e27217, 2011). With regard to DAPI-validated nuclear accumulation, Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining; Fig S1 panels Q, R, and S of Kale et al (2010) show DAPI-validated nuclear accumulation of AvrL567Nt-GFP, AvrLm6Nt-GFP and Avr2Nt-GFP respectively; Fig S2 panels B-D of Plett et al (Current Biology 21(14), 1197-12032011) show DAPI-validated nuclear localization of fluorescently labeled MiSSP7. I quote your response from our private email exchange: "Thanks for your response. It was very informative and did a lot to assuage many of my concerns. I need to digest the information a little more before I add comments to your document but I'm certainly a lot more convinced than I was previously. "
Brett Tyler's comment, April 26, 2013 6:57 PM
On a more general note, I'd like to state that this parlor game of plucking individual figures from papers, and trashing them, is not a constructive way to advance our field. Knowledge and understanding of complex problems are built step by step. It is NORMAL for publications that are released along the way to reveal many unresolved issues, some scientific and some technical. If we insist that all publications must be perfect in every way, and contain only figures that are perfect in every way, then NOTHING will get published, and progress will be greatly inhibited. Balanced, well thought out reviews (traditional pubs or blogs) that consider the advances as well as limitations of the recent literature as a whole are an appropriate and constructive vehicle to discuss where a field stands and where it needs to go next.
Mark Farman's comment, April 26, 2013 9:17 PM
Brett, At first your statement about the meristematic cells made me question whether my original criticisms of your data were valid. However, upon careful and in depth scrutiny of ALL of the published data and figures, I hold to my original criticism. Moreover, the new data raised even more serious concerns about your interpretations of the Avr1b data. I simply haven't had time to put my very long list of concerns down in writing. However, the basic issues are that: 1) NONE of the root uptake experiments for Avr1b have ever shown fluorescence at the cell periphery in a pattern consistent with cytoplasmic accumulation (your argument about meristematic cells not having a vacuole is irrelevant because most of the time, you are not looking at such cells). As an example, peripheral fluorescence is very clearly evident in root cells treated with Arg9-GFP (Dou et al 2008) and Ps87-GFP (Gu et al 2011) but this pattern was not obtained in parallel experiments with Avr1b RXLR. Even more convincing, however, were the plasmolysis data for Ps87 which clearly showed retraction of the fluorescence signals in concert with cytoplasmic shrinkage. Based on these images, I am almost convinced of the uptake of the Ps87 protein (my only reservation: missing controls to rule out autofluorescence). What I find most concerning, however, is that the very same figure presents data for Avr1b but corresponding plasmolysis data are conspicuously absent (and the pattern of fluorescence accumulation is completely different to that of Ps87). These cells are almost certainly dead, Brett - probably killed by high concentrations of Avr1b/Avr1bNt (and I believe this to be true for all the Avr1b/Avr1bNt assays). Until you present CONVINCING plasmolysis data to prove otherwise, your conclusions about Avr1b uptake into the cytoplasm will never hold water (showing "nuclear" uptake in the absence of plasmolysis data is insufficient for reasons I'll explain below). At present, the only published plasmolysis data for Avr1b (in the SUPPLEMENTARY data of Kale et al. 2010) is wholly unconvincing. I can't even tell what is going on in that image. Show me data for Avr1b that look like Gu et al 2011 Figure 4, panels F, G and I (along with controls to rule out autofluorescence), then I (and I imagine everyone else) will be satisfied that you have ruled out all of the alternative hypotheses that could explain your data (at least ones that satisfy Occum's razor).
2) Your GFP/mcherry uptake experiments have major issues:
i) you do not consider the relative quantum yields and photostabilities of GFP versus mcherry in your calculations. GFP has three times the quantum yield and twice the photostability of mcherry. That means when you see equal fluorescence intensities, mcherry is in ~6-fold molar excess over GFP.
ii) the densitometric scans across the root sections have been cherry-picked (excuse the pun) in a way that guarantees the results will support the hypothesis. On the very same root images, I can pick transects that will produce scans positively refuting it. You should be averaging the data across multiple transects laid at specific intervals along each individual root section (and then correcting the results to account for QY and photostability differences).
3) The Avr1b/Avr1bNt nuclear accumulation data are beset with major problems:
i) your Avr1bNt-GFP "nuclear" accumulation patterns are very unconvincing and have NEVER never been verified with DAPI (the example you give above is for Dylight- not GFP-labeled protein!).
ii) the Avr1b "nuclear" accumulation patterns show irregularly sized spots and patchy distribution which is VERY different to what I would expect to see and to what, in fact, was observed with Ps87 and the A9-GFP proteins. This highlights the critical need for DAPI confirmation.
iii) In Tyler et al (MPMI first look, 2013), Figure 2, panels G and J, green spots can be seen in cells that lack the expected peripheral cytoplasmic fluorescence (see Gu et al 2011). What we see, instead, is extensive fluorescence in the middle of the cell where the vacuole should be (note these are definitely NOT meristematic cells). In my lab, the only time we EVER see staining in this pattern is with dead cells that no longer plasmolyze. Thus, I can only conclude that the cells in panels G and J are dead or at least severely compromised - read "leaky"; and, in turn, I suspect that we are looking at nucleus "staining", as opposed to nuclear accumulation. Again, plasmolysis assays are needed.
iv) Finally, in your response to my critique, you stated that "Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining" Not true! According to the figure legend, these panels show nuclear accumulation of Avr1bNt-Dylight488! Explain that to me - does Dylight488 contain a nuclear uptake signal? If so, then why wasn't it taken up into the nuclei in panels B and E. Conversely, very nice green fluorescent spots show up in the bottom right-hand corner of panels B and E but these are NOT nuclei, as evidenced by the lack of DAPI staining. Major, major inconsistencies here Brett and, yet, you're only holding on to the data you want to see.
Hopefully, you realize that I am not plucking individual figures and papers. What I see are systemic holes in your data which means that my hypothesis (purified Avr1bNt-GFP kills soybean root cells, making them permeable to the protein) holds just as much water as yours.
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RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery | Direct but flimsy evidence for oomycete effector translocation | Scoop.it

RXLR-dEER-GFP Fusion Proteins Isolated from E. coli Can Enter Soybean Cells in the Absence of the Pathogen.
GFP fusion proteins were expressed in E. coli, partially purified, and incubated with soybean root tips for 12 h. The root tips were then washed for 4 h and photographed under UV and white light illumination.
(A) Protein gel electrophoresis analysis of GFP fusion proteins partially purified from E. coli cells: lane 1, Arg9-GFP; lane 2, GFP fused to the N-terminal 44 amino acids of mature wild-type Avr1b protein (RXLR1+,RXLR2+-dEER-GFP); lane 3, same as lane 2 with both RXLR1 and RXLR2 mutations (RXLR1AAAA,RXLR2AAAA-dEER-GFP); lane 4, same as lane 2 except with dEER mutation (RXLR1+,RXLR2+-dEERAAAAAA-GFP). The left lane contained molecular mass markers; the sizes of the markers are shown on the left (in kD). All expressed GFP proteins fluoresce normally under UV illumination.
(B) to (F) UV (left panels) and back-lit white light (right panels) illumination of roots after incubation with the indicated GFP protein fusion. The UV photographs represent longitudinal optical sections taken using the confocal microscope as illustrated by the dashed line in the inset of (C). The GFP concentration, illumination, and exposure of the UV photographs was identical in all 10 panels shown.
(B) Buffer alone with no fusion protein.
(C) RXLR1+,RXLR2+-dEER-GFP.
(D) Arg9-GFP.
(E) RXLR1AAAA,RXLR2AAAA-dEER-GFP.
(F) RXLR1+,RXLR2+-dEERAAAAAA-GFP.
(G) and (H) Higher-magnification photographs after the root tips were gently squashed following washing, showing nuclear accumulation of GFP.
(G) RXLR1+,RXLR2+-dEER-GFP.
(H) Arg9-GFP

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Brett Tyler's comment, April 26, 2013 6:44 PM
Mark, I'm surprised you haven't retracted these comments following our email exchanges. So I'm posting my rebuttal here. First, while leaf epidermal cells do have large vacuoles, root meristematic zone cells, and leaf mesophyll cells do NOT. This information is available in any plant cell biology text book, e.g. Biology 4th edition Ladiges et al. 2010. With regard to the vacuole, there is no evidence whatsoever for a continuous soluble phase connection between the apoplast and vacuole. This fact can also be found in the same text books. With regard to plasmolysis, Avr1bNt-GFP plasmolysis data were shown in Figure S1 panel O of Kale et al (Cell, 142(2), 284–295 2010). Similar plasmolysis data were shown for the fungal RxLR-like effector Ps87 in Figure 2 panel I of Gu et al (PLoS ONE 6(11), e27217, 2011). With regard to DAPI-validated nuclear accumulation, Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining; Fig S1 panels Q, R, and S of Kale et al (2010) show DAPI-validated nuclear accumulation of AvrL567Nt-GFP, AvrLm6Nt-GFP and Avr2Nt-GFP respectively; Fig S2 panels B-D of Plett et al (Current Biology 21(14), 1197-12032011) show DAPI-validated nuclear localization of fluorescently labeled MiSSP7. I quote your response from our private email exchange: "Thanks for your response. It was very informative and did a lot to assuage many of my concerns. I need to digest the information a little more before I add comments to your document but I'm certainly a lot more convinced than I was previously. "
Brett Tyler's comment, April 26, 2013 6:57 PM
On a more general note, I'd like to state that this parlor game of plucking individual figures from papers, and trashing them, is not a constructive way to advance our field. Knowledge and understanding of complex problems are built step by step. It is NORMAL for publications that are released along the way to reveal many unresolved issues, some scientific and some technical. If we insist that all publications must be perfect in every way, and contain only figures that are perfect in every way, then NOTHING will get published, and progress will be greatly inhibited. Balanced, well thought out reviews (traditional pubs or blogs) that consider the advances as well as limitations of the recent literature as a whole are an appropriate and constructive vehicle to discuss where a field stands and where it needs to go next.
Mark Farman's comment, April 26, 2013 9:26 PM
Brett, At first your statement about the meristematic cells made me question whether my original criticisms of your data were valid. However, upon careful and in depth scrutiny of ALL of the published data and figures, I hold to my original criticism. Moreover, the new data raised even more serious concerns about your interpretations of the Avr1b data. I simply haven't had time to put my very long list of concerns down in writing. However, the basic issues are that: 1) NONE of the root uptake experiments for Avr1b have ever shown fluorescence at the cell periphery in a pattern consistent with cytoplasmic accumulation (your argument about meristematic cells not having a vacuole is irrelevant because most of the time, you are not looking at such cells). As an example, peripheral fluorescence is very clearly evident in root cells treated with Arg9-GFP (Dou et al 2008) and Ps87-GFP (Gu et al 2011) but this pattern was not obtained in parallel experiments with Avr1b RXLR. Even more convincing, however, were the plasmolysis data for Ps87 which clearly showed retraction of the fluorescence signals in concert with cytoplasmic shrinkage. Based on these images, I am almost convinced of the uptake of the Ps87 protein (my only reservation: missing controls to rule out autofluorescence). What I find most concerning, however, is that the very same figure presents data for Avr1b but corresponding plasmolysis data are conspicuously absent (and the pattern of fluorescence accumulation is completely different to that of Ps87). These cells are almost certainly dead, Brett - probably killed by high concentrations of Avr1b/Avr1bNt (and I believe this to be true for all the Avr1b/Avr1bNt assays). Until you present CONVINCING plasmolysis data to prove otherwise, your conclusions about Avr1b uptake into the cytoplasm will never hold water (showing "nuclear" uptake in the absence of plasmolysis data is insufficient for reasons I'll explain below). At present, the only published plasmolysis data for Avr1b (in the SUPPLEMENTARY data of Kale et al. 2010) is wholly unconvincing. I can't even tell what is going on in that image. Show me data for Avr1b that look like Gu et al 2011 Figure 4, panels F, G and I (along with controls to rule out autofluorescence), then I (and I imagine everyone else) will be satisfied that you have ruled out all of the alternative hypotheses that could explain your data (at least ones that satisfy Occum's razor).
2) Your GFP/mcherry uptake experiments have major issues:
i) you do not consider the relative quantum yields and photostabilities of GFP versus mcherry in your calculations. GFP has three times the quantum yield and twice the photostability of mcherry. That means when you see equal fluorescence intensities, mcherry is in ~6-fold molar excess over GFP.
ii) the densitometric scans across the root sections have been cherry-picked (excuse the pun) in a way that guarantees the results will support the hypothesis. On the very same root images, I can pick transects that will produce scans positively refuting it. You should be averaging the data across multiple transects laid at specific intervals along each individual root section (and then correcting the results to account for QY and photostability differences).
3) The Avr1b/Avr1bNt nuclear accumulation data are beset with major problems:
i) your Avr1bNt-GFP "nuclear" accumulation patterns are very unconvincing and have NEVER never been verified with DAPI (the example you give above is for Dylight- not GFP-labeled protein!).
ii) the Avr1b "nuclear" accumulation patterns show irregularly sized spots and patchy distribution which is VERY different to what I would expect to see and to what, in fact, was observed with Ps87 and the A9-GFP proteins. This highlights the critical need for DAPI confirmation.
iii) In Tyler et al (MPMI first look, 2013), Figure 2, panels G and J, green spots can be seen in cells that lack the expected peripheral cytoplasmic fluorescence (see Gu et al 2011). What we see, instead, is extensive fluorescence in the middle of the cell where the vacuole should be (note these are definitely NOT meristematic cells). In my lab, the only time we EVER see staining in this pattern is with dead cells that no longer plasmolyze. Thus, I can only conclude that the cells in panels G and J are dead or at least severely compromised - read "leaky"; and, in turn, I suspect that we are looking at nucleus "staining", as opposed to nuclear accumulation. Again, plasmolysis assays are needed.
iv) Finally, in your response to my critique, you stated that "Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining" Not true! According to the figure legend, these panels show nuclear accumulation of Avr1bNt-Dylight488! Explain that to me - does Dylight488 contain a nuclear uptake signal? If so, then why wasn't it taken up into the nuclei in panels B and E. Conversely, very nice green fluorescent spots show up in the bottom right-hand corner of panels B and E but these are NOT nuclei, as evidenced by the lack of DAPI staining. Major, major inconsistencies here Brett and, yet, you're only holding on to the data you want to see.
Hopefully, you realize that I am not plucking individual figures and papers. What I see are systemic holes in your data which means that my hypothesis (purified Avr1bNt-GFP kills soybean root cells, making them permeable to the protein) holds just as much water as yours.